Styrene, isoprene, and 1,3-butadiene are important synthonsin the rubber and plastics industry. They have further utility in the production of epoxides which may be useful in a range of organic syntheses including the production pharmaceuticals. As waste products, these alkenes are released into the environment though natural processes, including the mineralization of plastics and rubbers, and as exhaust gas emissions resulting from the incomplete combustion of fossil fuels. Because of the widespread distributionof these compounds, and their reactivity as alkylating agents, there is growing concernand uncertainty regarding the risks of human exposure. For these reasons, significant research directed toward elucidation of the enzyme mechanisms involved in the processing of alkenes by humans and by microorganisms in the natural environment is warented. We will isolate, clone, express, and mechanistically characterize the two component syrene monoxgenase system from Pseudomonas putida (S12). Studies of the purified enzyme will be designed to elucidate the kinetics and thermodynamics of protein-protein, protein-substrate, and protein-coenzyme interactions as they pertain to the styrene epoxidation reaction. The steady state and pre-steady state kinetics of the oxidative and reductive half reactions will be analyzed by multiple wave length single and double mixing stopped-flow studies. Steps in the reductive half reaction corresponding to NADH-binding, FAD-binding, hydride-transfer, and the dissociation of NAD reduced FAD will be identified. The stereochemistry and deuterium isotope effects associted with hydride transfer will be established. Titrametric methods will be used to determine the redox potential of the bound-FAD and to establishany linkage between binding affinity and reduction state. Substrate analogs including isoprene, butadiene, and deuterated and halogenated styrenes will be used to probe the structure of the active site the nature of the oxygen reaction. Macroscopic pKa values of active site residuesinvolved in proton-transfer reactions will be established and steps involved in proton uptake or release will be identified. Activation energies associated with transition states in the oxygen and reduction reactions will be estimated by Eyring analysis.